hCas9

CRISPR Mouse - Deletion Dck

Experiment
CRISPR Mouse - Deletion Dck
Product
hCas9 from Addgene
Manufacturer
Addgene

Protocol tips

Protocol tips
There is no need to perform a negative control golden-gate reaction (without
insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

CRISPR knockouts
Candidate target sequences for CRISPR were designed using ZiFiT Targeter Version 4.2 (http://zifit.partners.org/ZiFiT/). The sequences of guide RNA were placed in pENTR221-U6-gRNA by inverse PCR, as previously described29. hCas9 was purchased from addgene (Plasmid #41815). hCas9 was PCR amplified and transferred to pENTE221 by standard BP Clonase reaction (Invitrogen), following manufacturers protocol. hCas9 was then transferred to PB-TRE-DEST-EF1A-rtTA-RES-Puro29 by standard LR Clonase reaction (Invitrogen), following manufacturers protocol, to generate PB-TRE-Cas9-EF1A-rtTA-IRES-Puro. The Gateway DEST cassette was then PCR amplified with NheI site engineered into the primers and cloned into a unique NheI site upstream of the TRE promoter to generate PB-DEST-TRE-Cas9-EF1A-rtTA-IRES-Puro. The guide RNAs were then transferred to PB-DEST-TRE-Cas9-EF1A-rtTA-IRES-Puro via standard LR Clonase reaction (Invitrogen), following manufacturers protocol. Two micrograms of each PB-U6-gRNA-TRE-Cas9-EF1A-rtTA-IRES-Puro and Super piggyBac transposase (System Biosciences) were transfected to B117P by NEON® Transfection System (Life Technologies Corporation, Carlsbad, CA) using 1,400 volts and 20 milliseconds for 2 pulses. Two days later transfected cells were selected with 1.5 μg/ml puromycin and 1.0 μg/ml of doxycycline for more than 3 weeks to generate stable knockout cell lines. DNA was collected using standard Phenol:Chloroform extraction. A CEL-I assay was performed (Supplementary Figure S5) and the gene modification ratio calculated, as previously described30.

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Reviews

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Discussion

Discussion

4 years ago

Author: Mario Udinese Italy

Floxing mice with CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Discussion

5 years ago

Author: Ben Saar Israel

How to choose a region to target for CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Share your thoughts or question with experts in your field by adding a discussion!

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Deletion Dck using hCas9 from Addgene.

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Manufacturer protocol

Download the product protocol from Addgene for hCas9 below.

Download PDF Download manufacturer protocol

Videos

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